Expression profiling of uterine leiomyomata cytogenetic subgroups reveals distinct signatures in matched myometrium: transcriptional profilingof the t(12;14) and evidence in support of predisposing genetic heterogeneity.

Uterine leiomyomata (UL), the most common neoplasm in reproductive-age women, are classified into distinct genetic subgroups based on recurrent chromosome abnormalities. To develop a molecular signature of UL with t(12;14)(q14-q15;q23-q24), we took advantage of the multiple UL arising as independent clonal lesions within a single uterus. We compared genome-wide expression levels of t(12;14) UL to non-t(12;14) UL from each of nine women in a paired analysis, with each sample weighted for the percentage of t(12;14) cells to adjust for mosaicism with normal cells. This resulted in a transcriptional profile that confirmed HMGA2, known to be overexpressed in t(12;14) UL, as the most significantly altered gene. Pathway analysis of the differentially expressed genes showed significant association with cell proliferation, particularly G1/S checkpoint regulation. This is consistent with the known larger size of t(12;14) UL relative to karyotypically normal UL or to UL in the deletion 7q22 subgroup. Unsupervised hierarchical clustering demonstrated that patient variability is relatively dominant to the distinction of t(12;14) UL compared with non-t(12;14) UL or of t(12;14) UL compared with del(7q) UL. The paired design we employed is therefore important to produce an accurate t(12;14) UL-specific gene list by removing the confounding effects of genotype and environment. Interestingly, myometrium not only clustered away from the tumors, but generally separated based on associated t(12;14) versus del(7q) status. Nine genes were identified whose expression can distinguish the myometrium origin. This suggests an underlying constitutional genetic predisposition to these somatic changes which could potentially lead to improved personalized management and treatment.

Incidence and patterns of ALK FISH abnormalities seen in a large unselected series of lung carcinomas.

BACKGROUND: Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements have been reported in 2-13% of patients with non-small cell lung cancer (NSCLC). Patients with ALK rearrangements do not respond to EGFR-specific tyrosine kinase inhibitors (TKIs); however, they do benefit from small molecule inhibitors targeting ALK. RESULTS: In this study, fluorescence in situ hybridization (FISH) using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in a large unselected cohort of 1387 patients with a referred diagnosis of non-small cell lung cancer (1011 of these patients had a histologic diagnosis of adenocarcinoma). The abnormal FISH signal patterns varied from a single split signal to complex patterns. Among 49 abnormal samples (49/1387, 3.5%), 32 had 1 to 3 split signals. Fifteen samples had deletions of the green 5' end of the ALK signal, and 1 of these 15 samples showed amplification of the orange 3' end of the ALK signal. Two patients showed a deletion of the 3'ALK signal. Thirty eight of these 49 samples (38/1011, 3.7%) were among the 1011 patients with confirmed adenocarcinoma. Five of 8 patients with ALK rearrangements detected by FISH were confirmed to have EML4-ALK fusions by multiplex RT-PCR. Among the 45 ALK-rearranged samples tested, only 1 EGFR mutation (T790M) was detected. Two KRAS mutations were detected among 24 ALK-rearranged samples tested. CONCLUSIONS: In a large unselected series, the frequency of ALK gene rearrangement detected by FISH was approximately 3.5% of lung carcinoma, and 3.7% of patients with lung adenocarcinoma, with variant signal patterns frequently detected. Rare cases with coexisting KRAS and EGFR mutations were seen.

Insertion (12;9)(p13;q34q34): a cryptic rearrangement involving ABL1/ETV6 fusion in a patient with Philadelphia-negative chronic myeloid leukemia.

We report a rare cryptic ins(12;9)(p13;q34q34), a chromosomal abnormality involving the ABL1 (9q34) and the ETV6 (alias TEL; 12p13) genes, detectable only by fluorescence in situ hybridization (FISH), in a patient with Philadelphia-negative chronic myeloid leukemia (CML). Using reverse 4',6-diamidino-2-phenylindole banding on metaphase cells, FISH analysis with BCR/ABL dual-fusion and ETV6 break-apart probes showed that a third ABL signal was inserted into 12p, splitting the ETV6 signal into two adjacent signals. CML patients with an ABL1/ETV6 fusion historically have demonstrated a variable and sometimes transient response to treatment with imatinib mesylate, which was also the case in the present patient.

Uterine leiomyomata with deletions of Ip represent a distinct cytogenetic subgroup associated with unusual histologic features.

Cytogenetic analysis of uterine leiomyomata (UL) shows that about 40% of these benign tumors have simple, clonal chromosomal rearrangements. In contrast, their presumed malignant counterpart, leiomyosarcomas (LMSs), typically has complex numerical and structural abnormalities. Several variants of benign uterine smooth-muscle tumors are defined by histologic phenotypes intermediate between typical UL and LMS, and currently, little is known about their cytogenetic and molecular genetic features. From a subset of more than 800 karyotyped ULs, we identified a group of nine cases exhibiting near-diploid karyotypes with loss of almost the entire short (p) arm of chromosome 1 [i.e., del(1)(p11p36)]. Loss of 1p was often associated with other aberrations, particularly loss of chromosomes 19 and/or 22. Of eight UL for which the histologic diagnosis was known, four were diagnosed as cellular UL; one displayed both hypercellularity and nuclear atypia. Loss of heterozygosity (LOH) analysis for chromosomal regions 1p36.23 and 1p21.1 demonstrated allelic loss for either a portion or the majority of 1p in 5 of 10 additional archival UL diagnosed with either cellular or atypical histology. RNA from two UL with loss of 1p was profiled using Affymetrix GeneChips, and those profiles were compared to our previously reported smooth-muscle tumor expression profile. The transcriptional profiles of tumors with 1p deletion were more similar to those of leiomyosarcoma than to profiles of myometrium and UL, as determined by hierarchical cluster analysis. Comparison of the transcriptional profiles for UL with and without 1p-- revealed 53 genes with differential regulation. Loss of 1p appears to define a subgroup of UL distinct from those previously recognized. Furthermore, 1p-- appears to be associated with a specific histologic phenotype. The similarity between the transcriptional profiles of LMS and UL with 1p-- suggests the possibility of a common pathogenetic mechanism.

Impact of patient mutations on heterodimer formation and function in human galactose-1-P uridylyltransferase.

Impairment of the human enzyme galactose-1-P uridylyltransferase (hGALT) results in the potentially lethal disorder, galactosemia. One of the fundamental questions with regard to this dimeric enzyme involves the possible influence of patient mutations on heterodimer formation and activity. Indeed, considering that many if not most galactosemia patients are compound heterozygotes, this is an issue of clinical as well as basic science interest. We have utilized a yeast expression system for the human enzyme to test whether each of a small number of mutations in hGALT (S135L, F171S, F171W, Q188R, N314D, and R333W) impact either heterodimer formation or function. Our results clearly demonstrate that while a majority of the alleles tested show precisely random patterns of subunit assortment, two deviate slightly but significantly from this pattern. Similarly, while some heterodimers exhibit apparent independence of subunit activity, others do not. These data not only demonstrate that common patient mutations in hGALT can influence both heterodimer formation and function in heterozygotes, they further raise the question of whether such interactions may also occur between different mutant alleles in compound heterozygotes (i.e., patients). Indeed, such influences may underlie some of the biochemical and clinical heterogeneity observed in the galactosemia patient population.